Chemicals and plastic particles
All chemicals were purchased from Merck (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Sigma-Aldrich (Taufkirchen, Germany) if not stated otherwise. The 366 nm MF particles “MF366” (charge: MF-FluoOrange-S886–1, Ex/Em 560 nm/584 nm) were purchased from Microparticles GmbH (Berlin, Germany), both PLA particle types (250 nm and 2 μm) “PLA250” and “PLA2000” (PLA-greenF, prod.-no: 51–00-252 and 51–00-203, Ex/Em 502 nm/527 nm) were purchased from Micromod particle technology GmbH (Rostock, Germany) and 25 nm PMMA particles “PMMA25” (DiagPoly™ Plain Fluorescent PMMA nanoparticles, cat.-no.: DNG-P010) were purchased from Creative Diagnostics (New York, USA). The 10 μm PS particles (prod.-no.: PFH-10056, Ex/Em 530 nm/582 nm) were bought from Kisker Biotech GmbH (Steinfurt, Germany), the 4 μm PS particles (prod.no.: 2219, Ex/Em 530 nm/582 nm) from Phosphorex Inc. (United States) and the 1 μm PS particles (prod.no.: F13080, Ex/Em 505 nm/515 nm) were purchased from life technologies (Carlsbad, Germany). When investigating MNPs, it is important to distinguish between effects caused by the material, dispersant, the size and/or the surface. Especially the dispersant can play a role in evoking adverse effects on cells, which underlines the importance of having an adequate set of controls [25]. To ensure any dispersant-related effects were excluded in this study, all particles were delivered as aqueous dispersions. An overview about the purchased plastic particles and the applied methods can be found in the supporting information (Table S1).
Characterization of particles
Plastic particles were characterized using Fluorescence Microscopy, Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Asymmetric Flow Field Flow Fractionation (AF4), Small-Angle X-ray Scattering (SAXS) and assessing an octanol-water distribution coefficient (hydrophobicity).
Fluorescence microscopy
To check for particle size, shape and fluorescence signal, stock plastic dispersions were analyzed in their stock concentrations using the inverse microscope axio observer d1 (Carl Zeiss, Oberkochen, Germany). Images were recorded using Brightfield, EGFP (Ex/Em 488/509) or Cy5 (Ex/Em 650/673 nm) filter depending on the wavelength of the particles label.
SEM
For precise verification of particle sizes and shape, pristine plastic dispersions were analyzed using a Zeiss DSM 982 Gemini (Carl Zeiss AG, Oberkochen, Germany; updated by point electronic GmbH, Halle (Saale), Germany) conducted with an acceleration voltage between 6 and 9 kV as described in a study using microplastics before [23]. Size distribution was quantified on the basis of the SEM images by using the ImageJ software V.1.53 (Laboratory for Optical and Computational Instrumentation (LOCI) of the University of Wisconsin-Madison, Madison, USA). The Feret diameter was determined by using the function ‘Measure’ and measuring at least 200 random particles per type. The scale bar of the SEM images was used to define a scale in the program. Results are shown as histograms.
DLS
Hydrodynamic diameter and zeta potential were measured with a Zetasizer Nano ZS (Malvern Panalytical GmbH, Kassel, Germany). Plastic particles were diluted 1:1000 in ultrapure water and subsequently measured. Results for hydrodynamic diameters are given as size distribution curves and polydispersity indices. For zeta potential, the universal ‘Dip’ Cell Kit (Malvern Panalytical GmbH, Kassel, Germany) was used. Mean values and standard deviations of at least three measurements were calculated. Since experiments were performed in cell culture media, further DLS measurements in medium at 20 °C and 37 °C and for incubation time points of 0 h and 24 h were performed.
AF4
Particle sizes were further determined by AF4 coupled to Multi Angle and Dynamic Light Scattering (MALS/DLS) for all particle types except of PLA2000. The AF4 system consisted of an Agilent 1200 series autosampler (G1329A), a high-performance liquid chromatography pump (G1311A) (Agilent Technologies, Santa Clara, CA, USA), an Eclipse 3 AF4 flow control module, and a short channel-type AF4 separation channel with a 350 μm spacer (Wyatt Technology Europe GmbH, Dernbach, Germany). The carrier liquid was ultrapure water containing the alkaline detergent mix Fisherbrand FL-70 (Fisher Scientific, Pittsburgh, MA, USA) at a concentration of 0.025% (v/v) or ReagentPlus sodium dodecyl sulfate (SDS, Sigma Aldrich, St. Louis, MO, USA) at a concentration of 0.05% (m/v). Following separation by AF4, a DAWN HELEOS TM (Wyatt Technology Europe GmbH, Dernbach, Germany) MALS detector with 17 observation angles operated with a linear polarized laser light at 658 nm was used to record the light scattering signal. The data collection interval was set to 2 s. A DLS detector at angle 99° of the DAWN HELEOS light scattering cell was used for on-line determination of the hydrodynamic diameter dh of the particles with an interval of 1 s. Data from the light scattering detectors was processed using the ASTRA V software (version 5.3.4.20, Wyatt Technology Corporation, Santa Barbara, CA, USA). The MALS detector at angle 90° was used for light scattering detection of the particles. The root mean square (rms) diameter drms was determined using a 3rd order Debye model because of its robustness and fitting capabilities for both spherical and non-spherical particles [26]. Before analysis, the samples were diluted in carrier liquid 1:500 (PLA250, MF366) or 1:50 (PMMA25). The instrumental settings are presented in Table S2.
SAXS
The goal of the SAXS measurements was to determine the size distribution of the particles in situ. Measurements were performed in a flow through capillary with the Kratky-type instrument SAXSess (Anton Paar, Graz, Austria) at 21 ± 1 °C. The SAXSess has a low sample-to-detector distance of 0.309 m, which is appropriate for investigation of dispersions with low scattering intensities. The samples were measured as delivered. The scattering vector q depends on the wavelength λ of the radiation (λ = 0.154 nm): thus q = 4π / λ sinθ. Deconvolution (slit length desmearing) of the SAXS curves was performed with the SAXS-Quant software. Curve fitting was conducted with software SASfit as described earlier [27]. The analysis of the SAXS raw data was performed using forms presented in the supporting information.
Hydrophobicity
To elucidate the distribution of plastic particles in hydrophilic or lipophilic phases and exclude experimental leaching effects, an octanol-water-distribution was assessed. Respective plastic particles were diluted 1:10 in ultrapure water, mixed in equal amounts with octanol and vortexed for 10 sec. Phases were separated again by shortly spinning the dispersion in a table centrifuge. The content of particles in the hydrophilic and lipophilic phase was measured using the Tecan plate reader (Plate Reade Infinite® M200Pro. Tecan Group Ltd., Männedorf, Switzerland). Another 1:2 dispersion of diluted particles with octanol was mixed for 24 h at room temperature. The fluorescence intensity of the phases was measured again. The two phases were ultracentrifuged at 186000 x g for 40 min and the fluorescence of the supernatant measured, but no significant fluorescence was detected (data not shown).
Cell cultivation
Caco-2 (ECACC: 86010202) and HepG2 (ECACC: 85011430) were purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK). HepaRG cells were obtained from Biopredic International (Saint Grégoire, France). The cells were cultivated as published before [23, 28]. Cultivation of cells was performed at 37 °C and 5% CO2. Caco-2 and HepG2 were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Pan-Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal calf serum superior (FCS superior), 105 Units/L penicillin and 100 μg/mL streptomycin (P/S; Capricorn Scientific GmbH, Ebsdorfergrund, Germany). HepaRG cells were cultured in Williams E medium (Pan-Biotech GmbH, Aidenbach, Germany) supplemented with 10% FCS (Pan-Biotech GmbH, Aidenbach, Germany, 1% P/S, 0.05% of 100 μg/ml human insulin (PAA Laboratories GmbH, Pasching, Austria), and 50 μM hydrocortisone-hemisuccinate (Sigma-Aldrich, Taufkirchen, Germany). These cells were passaged every 2 weeks. Caco-2 and HepG2 were passaged every 2–3 days at 80–90% confluence. This was conducted by aspirating the cell culture medium, washing the cells with phosphate-buffered saline (PBS) and subsequently incubating them with 0.05% of trypsin-ethylenediaminetetraacetic acid (EDTA) at 37 °C for 5 min (Caco-2) or 7 min (HepG2). Followed by adding 8.5 ml cell culture medium and centrifuging the cell suspension. The cell pellet was resuspended in fresh cell culture medium.
Cell viability testing
Caco-2, HepG2 and HepaRG cells were seeded in 96-well plates with concentrations of 5000 (Caco-2), 20,000 (HepG2) and 9000 (HepaRG) cells per well in their respective cell culture medium. HepG2 cells can be used for experiments 24 h after seeding. Caco-2 and HepaRG cells were differentiated for 3 weeks and 4 weeks, respectively. The HepaRG cells were cultivated in their cell culture medium for 2 weeks and further differentiated for another 2 weeks by adding 1.7% Dimethylsulfoxide (DMSO, v/v) to the proliferation medium. To perform the incubation, cell culture medium was replaced by 100 μL phenol red-free cell culture medium containing different concentrations of plastic particles. The maximum concentration was chosen to exclude effects due to nutrient deficiency of cells, while at the same time ensuring concentrations would result in toxic effects being observed. The unit μm2 particle surface/mL was used to apply concentrations with comparable particle surface area exposed to the cells and to therefore exclude toxic effects based on the increasing surface-to-volume ratios of the smaller particles. To achieve comparability, the resulting concentrations in particles/mL and the conversion factor are presented in Table S3. After 24 h or 48 h of incubation, the particle dispersions were aspirated and substituted by 100 μl phenol red-free medium and the 3-(4,5-dimethylthioazole-2-zyl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed, as previously described [23], to determine the metabolic activity of cells after incubation with plastic particles. A 5 mg/mL solution of MTT in PBS was thus added at a volume of 10 μL per well and incubated for 1 h. The supernatant was removed afterwards and 130 μL per well of a desorption solution containing 0.7% (w/v) SDS in isopropanol was added. The 96-well plates were shaken for another 30 min under light exclusion to dissolve the formazan crystals. Absorption was measured at 570 nm and 630 nm background absorption. A concentration 0.01% Triton X-100 was used as positive control. To evaluate the results, raw data were subtracted by background signals (wells that were incubated with corresponding particle concentrations and all assay components, but did not contain cells and the medium control). This value was related to the solvent control (cells incubated with cell culture medium, but no particles), which was set to 100%. Mean values and standard deviations were calculated for at least three independent experiments.
To test the cell viability for longer incubation times, the xCELLigence® Real Time Cell Analysis (Agilent Technologies Germany GmbH & Co. KG, Waldbronn, Germany) was used on the basis of a protocol published earlier [29]. Caco-2, HepG2 or HepaRG cells were seeded as explained above on special 96-well microplates, coated with gold microelectrodes. Cells were differentiated and incubated with 150 μL of particle dispersions in cell culture medium diluted to different concentrations (ranging from 5 × 107–2.5 × 1010 μm2 particle surface/mL. The cell index was measured every hour for 72 h in total. 50 μg/mL zinc oxide (ZnO) served as positive control.
Particle uptake and transport through the intestinal barrier and uptake in hepatic cells
Intestinal barrier
To verify the particle uptake and transport through the intestinal epithelium, 12-well Transwell® plates consisting of polycarbonate membrane inserts with 1.12 cm2 growth area and 3 μm pore size (Corning Incorporated, New York, USA) were used, based on a protocol established by Stock et al. [23]. Short, 50,000 Caco-2 cells were seeded on top of the membrane of the Transwell® inserts and differentiated for 3 weeks. The cell culture medium was changed every two or three days. To check for the permeability of the monolayer of Caco-2 cells, transepithelial electrical resistance (TEER) measurements and transport of fluorescein isothiocyanate-dextran (FITC) were applied. Permeability values > 700 Ω*cm2 (TEER) and < 107 cm/s (PAPP values calculated from FITC transport) demonstrated the overall integrity of the Caco-2 monolayer. After three weeks of differentiation, cells were incubated with high, but non-toxic concentration (determined beforehand by cell viability tests) of plastic particles for 24 h in the apical compartment or with cell culture medium as a control. This was performed by removing the cell culture medium and replacing by a 500 μl particle dispersion that was diluted to a concentration of 2.5 μm2x109 μm2 particle surface/mL in the apical side and 500 μl cell culture medium without particles in the basolateral side. Afterwards, the apical and basolateral, as well as washing fractions (2 × 250 μL PBS), were collected and fluorescence intensity for each particle type measured at the Tecan plate reader. To be able to quantify the signals, calibration curves of the particles in respective medium (cell culture medium or PBS) were prepared and measured with the Tecan plate reader. The highest selected concentration was the initial particle concentration used for incubation. The Transwell® membranes were fixed with 3.7% formaldehyde solution in PBS for 30 min at 37 °C. In the next step, the fixed membranes were washed three times with PBS and the whole membrane was scanned with the Tecan plate reader to determine the fluorescence signals present in the membranes. By adding known amounts of the particle concentrations used for incubation and measuring the increase of the fluorescence, a standard curve was calculated. Afterwards, membranes were washed again three times with PBS.
To examine the interaction (comprising cellular uptake and adsorption) of the particles with the Caco-2 cells, a confocal microscope (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) was used. The fixed and washed Transwell® membranes were further prepared by permeabilization with 0.2% Triton-X 100 in PBS for 20 min. After washing for three times with PBS, the cells were stained with 2 drops/mL ActinGreen 488 ReadyProbes® Reagent (wells incubated with MF particles) or 2 drops/mL ActinRed 555 ReadyProbes® Reagent (wells incubated with PLA or PMMA particles; Life technologies, New York, USA) for 30 min under light exclusion. In the last step, the membranes were washed three times with PBS and the insert was cut off by using a scalpel. The membrane was fixed on a microscope slide by using Kaiser’s glycerin gelatin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and cover glasses. The samples were dried over night at 4 °C and examined on the next day. For each particle type, five random sections of the membrane from 2 to 3 replicates per particle were investigated. Due to resolution limits of the confocal microscope, sections were not quantified. Images were recorded by using the XYZ acquisition mode and the contrast was adjusted. This shows the membrane from its lateral side starting from the villi of the cells and ending at the membrane.
Hepatic cells
To check for uptake of plastic particles into hepatic cells, fluorescence microscopy was used with HepG2 and HepaRG cells [29]. Since HepG2 are proliferating hepatocyte-like cells without the capability of expressing enzymes of the xenobiotic metabolism and HepaRG are differentiated bilary- and hepatocyte-like cells expression Phase I and Phase II enzymes, a comparison of different uptake based on the cell models complexity. Therefore, HepG2 or differentiated HepaRG cells were incubated with high, but non-toxic concentrations of respective MNPs for 24 h. Cells were washed twice with PBS to remove particles laying on top of the cells. For microscopic examinations, cells were covered with PBS and the uptake of particles was assessed using an Axio Observer D1 (Carl Zeiss, Oberkochen, Germany). Images were assessed using Bright, EGFP and Cy5 filter dependent on the particles label.
Flow cytometry
To establish a flow cytometry-based method to study uptake of plastic particles in liver cells, only HepG2 cells were used due to their easier handling in comparison to HepaRG cells. The protocol was based on a previously published study [29]. The efficacy of the method was evaluated by characterizing the uptake of previously studied PS particles in the micrometer size range (10 μm, 4 μm, 1 μm) and at concentrations of 2.5 × 108 μm2 particle surface/mL, and then applied to the other test particles used in this study. Differentiated cells were incubated for 2, 4, 6 and 24 h, respectively to trace the increase of particle uptake with time at concentrations of 5 × 108 μm2 (PLA2000), 2.5 × 109 (PLA250), 2.5 × 108 μm2 (MF366) and 2 × 1011 (PMMA25) particle surface/mL, dependent on the particles toxicity on HepG2 cells. After washing the cells twice with PBS, the incubated cells were subsequently harvested using trypsin-EDTA. The cell suspension was centrifuged at 300 x g for 5 min, resuspended in 100 μL PBS and used for measurements. To do so, the flow cytometer BD Accuri C6 (BD, Heidelberg, Germany) was utilized with an excitation laser at 488 nm. The optical filters FL1 (533/30 nm), FL2 (585/40 nm) or FL3 (> 670 nm) were used dependent on the fluorescence of the particles. Signals were detected for 2 min or a maximum of 10,000 cells. For analysis, the cell population was identified by using the side and forward scatter Histogram and a Gate P1 was set. Only signals in this gate were further used for quantification. The uptake of particles into cells was measured by using the fluorescence signal normalized to the intrinsic signal of the cells. Furthermore, a side scatter analysis was done to check for increased granularity. Granularity is a measure of the inner structure of cells, whereby a high granularity, indicated by high side scatter values, indicates structural inclusions. Therefore, a side scatter analysis is often used to indicate particle uptake into cells [29, 30].
Statistical analysis
Statistical analysis was performed using SigmaPlot 14.0 (Systat Software GmbH, Erkrath, Germany). For cytotoxicity assays, one-way ANOVA following Dunnett’s test was used, thereby comparing untreated medium controls with cells that were treated with respective nano- or microplastic particles. Statistical analysis as well as means and standard deviations were performed for at least three independent experiments. The significance levels were defined by performing one-way ANOVA Dunnett’s test and calculating p-values (* = p < 0.05, ** = p < 0.01, *** = p ≤ 0.001).